DNA Methylation And Its Basic Function
REVIEWNeuropsychopharmacology REVIEWS (2012), 1–16&2012 American College of Neuropsychopharmacology. All rights reserved 0893-133X/12.www.neuropsychopharmacology.orgDNA Methylation and Its Basic FunctionLisa D Moore1, Thuc Le1 and Guoping Fan*,11Interdepartmental Program in Neuroscience and Department of Human Genetics, David Geffen School of Medicine,University of California, Los Angeles, Los Angeles, CA, USAIn the mammalian genome, DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5position of the cytosine to form 5-methylcytosine. DNA methylation regulates gene expression by recruiting proteins involvedin gene repression or by inhibiting the binding of transcription factor(s) to DNA. During development, the pattern of DNAmethylation in the genome changes as a result of a dynamic process involving both de novo DNA methylation anddemethylation. As a consequence, differentiated cells develop a stable and unique DNA methylation pattern that regulatestissue-specific gene transcription. In this chapter, we will review the process of DNA methylation and demethylation in thenervous system. We will describe the DNA (de)methylation machinery and its association with other epigenetic mechanismssuch as histone modifications and noncoding RNAs. Intriguingly, postmitotic neurons still express DNA methyltransferasesand components involved in DNA demethylation. Moreover, neuronal activity can modulate their pattern of DNA methylationin response to physiological and environmental stimuli. The precise regulation of DNA methylation is essential for normalcognitive function. Indeed, when DNA methylation is altered as a result of developmental mutations or environmental riskfactors, such as drug exposure and neural injury, mental impairment is a common side effect. The investigation into DNAmethylation continues to show a rich and complex picture about epigenetic gene regulation in the central nervous system andprovides possible therapeutic targets for the treatment of neuropsychiatric disorders.Neuropsychopharmacology Reviews advance online publication, 11 July 2012; doi:10.1038/npp.2012.112Keywords: epigenetics; gene regulation; neuron; synaptic plasticity; demethylationINTRODUCTIONGenetics is the study of heritable changes in gene activity orfunction due to the direct alteration of the DNA sequence.Such alterations include point mutations, deletions, insertions, and translocation. In contrast, epigenetics is the studyof heritable changes in gene activity or function that is notassociated with any change of the DNA sequence itself.Although virtually all cells in an organism contain thesame genetic information, not all genes are expressedsimultaneously by all cell types. In a broader sense,epigenetic mechanisms mediate the diversified gene expression profiles in a variety of cells and tissues in multicellularorganisms.In this chapter, we would introduce a major epigeneticmechanism involving direct chemical modification to theDNA called DNA methylation. Historically, DNA methylation was discovered in mammals as early as DNA was*Correspondence: Dr G Fan, Interdepartmental Program in Neuroscienceand Department of Human Genetics, David Geffen School of Medicine,University of California, Los Angeles, 695 Charles Young Drive South, LosAngeles, CA 90095, USA, Tel: 1 310 267 0439, Fax: 1 310 794 5446,E-mail: gfan@mednet.ucla.eduReceived 6 March 2012; revised 7 May 2012; accepted 8 May 2012identified as the genetic material (Avery et al, 1944; McCartyand Avery, 1946). In 1948, Rollin Hotchkiss first discoveredmodified cytosine in a preparation of calf thymus usingpaper chromatography. Hotchkiss (1948) hypothesized thatthis fraction was 5-methylcytosine (5mC) because itseparated from cytosine in a manner that was similar tothe way that thymine (also known as methyluracil) separatedfrom uracil, and he further suggested that this modifiedcytosine existed naturally in DNA. Although many researchers proposed that DNA methylation might regulate geneexpression, it was not until the 1980s that several studiesdemonstrated that DNA methylation was involved in generegulation and cell differentiation (Holliday and Pugh, 1975;Compere and Palmiter, 1981). It is now well recognized thatDNA methylation, in concert with other regulators, is amajor epigenetic factor influencing gene activities.DNA methylation is catalyzed by a family of DNAmethyltransferases (Dnmts) that transfer a methyl groupfrom S-adenyl methionine (SAM) to the fifth carbon of acytosine residue to form 5mC (Figure 1). Dnmt3a andDnmt3b can establish a new methylation pattern tounmodified DNA and are thus known as de novo Dnmt(Figure 1a). On the other hand, Dnmt1 functions duringDNA replication to copy the DNA methylation pattern from.Neuropsychopharmacology REVIEWS1
DNA methylation and its basic functionLD Moore et alREVIEW.2the parental DNA strand onto the newly synthesizeddaughter strand (Figure 1b). All three Dnmts are extensivelyinvolved in the development of an embryo. By the time cellsreach terminal differentiation, Dnmt expression is muchreduced. This would seem to suggest that the DNA methylation pattern in postmitotic cells is stable. However,postmitotic neurons in the mature mammalian brain stillexpress substantial levels of Dnmts, raising the possibilitythat Dnmts and DNA methylation may play a novel role inthe brain (Goto et al, 1994; Feng et al, 2005).Neurons react to the environment through patterns ofdepolarization that both relay information and encode aresponse. In recent years, it has become increasinglyapparent that following depolarization, alterations in geneexpression are accompanied by modifications of theepigenetic landscape that include alterations in the patternof DNA methylation (Martinowich et al, 2003; Guo et al,2011a). In order for the DNA methylation pattern to bealtered, there must be both active DNA methylation anddemethylation in the neuronal genome. However, noenzymes are known to cleave the methyl group directlyfrom 5mC. As discussed below, the recent identification of5-hydroxymethyl-cytosine (5hmC) in postmitotic neuronssuggests that 5hmC serves as an intermediate in the DNAa3′5′3′A AC TGT TCTCCCCGGGGCAAT 3′GCT 3′G5′5′Figure 1. DNA methylation pathways. A family of DNA methyltransferases (Dnmts) catalyzes the transfer of a methyl group from S-adenylmethionine (SAM) to the fifth carbon of cytosine residue to form 5methylcytosine (5mC). (a) Dnmt3a and Dnmt3b are the de novo Dnmtsand transfer methyl groups (red) onto naked DNA. (b) Dnmt1 is themaintenance Dnmt and maintains DNA methylation pattern duringreplication. When DNA undergoes semiconservative replication, theparental DNA stand retains the original DNA methylation pattern (gray).Dnmt1 associates at the replication foci and precisely replicates theoriginal DNA methylation pattern by adding methyl groups (red) onto thenewly formed daughter strand (blue).Neuropsychopharmacology REVIEWSAlthough the brain contains some of the highest levels ofDNA methylation of any tissue in the body, 5mC onlyaccounts for B1% of nucleic acids in the human genome(Ehrlich et al, 1982). The majority of DNA methylationoccurs on cytosines that precede a guanine nucleotide orCpG sites. Overall, mammalian genomes are depleted ofCpG sites that may result from the mutagenic potential of5mC that can deaminate to thymine (Coulondre et al, 1978;Bird, 1980). The remaining CpG sites are spread out acrossthe genome where they are heavily methylated with theexception of CpG islands (Bird et al, 1985). Interestingly,there is evidence of non-CpG methylation in mouse andhuman embryonic stem cells, however these methylationare lost in mature tissues (Ramsahoye et al, 2000; Listeret al, 2009). More thorough analysis of the murine frontalcortex has recently revealed that although the majority ofmethylation occurs within CpG sites, there is a significantpercentage of methylated non-CpG sites (Xie et al, 2012).Because of its recent discovery, the role of non-CpGmethylation is still unclear.DNA methylation is essential for silencing retroviralelements, regulating tissue-specific gene expression, genomic imprinting, and X chromosome inactivation. Importantly, DNA methylation in different genomic regions mayexert different influences on gene activities based on theunderlying genetic sequence. In the following sections, wewill further elaborate upon the role of DNA methylation indifferent genomic regions.Intergenic RegionsTCATGT5′LOCATION OF DNA METHYLATIONCH3CAGCTTGCADnmt1A3′A 5′TGCCGCGACATGT5′GGCCCH33′T TGACAGCCGT5′3′A AC TGT CGGCAbDnmt3aDnmt3bA 5′3′T TGACAGCCGTT5′CH3demethylation pathway. In this review, we will discuss thebasic function of DNA methylation in epigenetic generegulation, and further highlight its role in neural development and neurological disease.Approximately 45% of the mammalian genome consists oftransposable and viral elements that are silenced by bulkmethylation (Schulz et al, 2006). The vast majority of theseelements are inactivated by DNA methylation or bymutations acquired over time as the result of the deamination of 5mC (Walsh et al, 1998). If expressed, these elementsare potentially harmful as their replication and insertioncan lead to gene disruption and DNA mutation (Michaudet al, 1994; Wu et al, 1997; Kuster et al, 1997; Gwynn et al,1998; Ukai et al, 2003). The intracisternal A particle (IAP) isone of most aggressive retroviruses in the mouse genome(Walsh et al, 1998). IAP is heavily methylated throughoutlife in gametogenesis, development, and adulthood (Walshet al, 1998; Gaudet et al, 2004). Even within the embryowhen the rest of the genome is relatively hypomethylated,Dnmt1 maintains the repression of IAP elements (Gaudetet al, 2004). When Dnmt1 is depleted by genetic mutations,leading to extensive hypomethylation, IAP elements areexpressed (Walsh et al, 1998; Hutnick et al, 2010). This
REVIEWDNA methylation and its basic functionLD Moore et al.3demonstrates that within intergenic regions, one of themain roles of DNA methylation is to repress the expressionof potentially harmful genetic elements.CpG IslandsCpG islands are stretches of DNA roughly 1000 base pairslong that have a higher CpG density than the rest of thegenome but often are not methylated (Bird et al, 1985). Themajority of gene promoters, roughly 70%, reside within CpGislands (Saxonov et al, 2006). In particular, the promoters forhousekeeping genes are often imbedded in CpG islands(Gardiner-Garden and Frommer, 1987). CpG islands, especially those associated with promoters, are highly conservedbetween mice and humans (Illingworth et al, 2010). Thelocation and preservation of CpG islands throughoutevolution implies that these regions possess a functionalimportance.It appears that CpG islands have been evolutionarilyconserved to promote gene expression by regulating thechromatin structure and transcription factor binding. DNAis regularly wrapped around histone proteins forming small,packaged sections called nucleosomes. The more tightlyassociated with histone proteins the DNA is, the lesspermissive it is for gene expression. One of the commonfeatures of CpG islands is that they contain less nucleosomes than other stretches of DNA (Tazi and Bird, 1990;Ramirez-Carrozzi et al, 2009; Choi, 2010). The fewnucleosomes with which CpG islands are associated oftencontain histones with modifications involved in enhancinggene expression (Tazi and Bird, 1990; Mikkelsen et al,2007). Although B50% of CpG islands contain knowntranscription start sites, CpG islands are often devoid ofcommon promoter elements such as TATA boxes (Carninciet al, 2006). As many transcription factor binding sites areGC rich, CpG islands are likely to enhance binding totranscriptional start sites. Despite their lack of commonpromoter elements, CpG islands enhance the accessibility ofDNA and promote transcription factor binding.The methylation of CpG islands results in stable silencingof gene expression (Mohn et al, 2008). During gametogenesisand early embryonic development, CpG islands undergodifferential methylation (Wutz et al, 1997; Caspary et al,1998; Zwart et al, 2001; Kantor et al, 2004). The ability ofmethylation to regulate gene expression through CpGislands is particularly important for establishing imprinting(Wutz et al, 1997; Caspary et al, 1998; Zwart et al, 2001; Choiet al, 2005). Imprinted genes are expressed from only one ofthe two inherited parental chromosomes and their expression is determined by the parent of inheritance. Beyondimprinted genes, DNA methylation of CpG islands regulatesgene expression during development and differentiation(Shen et al, 2007; Weber et al, 2007; Fouse et al, 2008; Mohnet al, 2008; Meissner et al, 2008). As CpG islands areassociated with the control of gene expression, it would beexpected that CpG islands might display tissue-specificpatterns of DNA methylation. Although CpG islands inintragenic and gene body regions can have tissue-specificpatterns of methylation, CpG islands associated withtranscription start sites rarely show tissue-specific methylation patterns (Rakyan et al, 2004; Eckhardt et al, 2006;Meissner et al, 2008; Illingworth et al, 2010; Maunakea et al,2010). Instead, regions called CpG island shores, located asfar as 2 kb from CpG islands, have highly conserved patternsof tissue-specific methylation (Irizarry et al, 2009). Like CpGislands, the methylation of CpG shores is highly correlatedwith reduced gene expression (Irizarry et al, 2009).The role of CpG islands in regulating gene expression isstill being uncovered. Methylation of CpG islands canimpair transcription factor binding, recruit repressivemethyl-binding proteins, and stably silence gene expression. However, CpG islands, especially those associated withgene promoters, are rarely methylated. Further studies areneeded to determine to what degree DNA methylation ofCpG islands regulates gene expression.Gene BodyAs the majority of CpG sites within the mammalian genomeare methylated, the genes themselves must also containmethylation. The gene body is considered the region of thegene past the first exon because methylation of the firstexon, like promoter methylation, leads to gene silencing(Brenet et al, 2011). Evidence suggests that DNA methylation of the gene body is associated with a higher level ofgene expression in dividing cells (Hellman and Chess, 2007;Ball et al, 2009; Aran et al, 2011). However, in slowlydividing and nondividing cells such as the brain, gene bodymethylation is not associated with increased gene expression (Aran et al, 2011; Guo et al, 2011a, b; Xie et al, 2012).Furthermore, in the murine frontal cortex, methylation ofnon-CpG sites within gene bodies is negatively correlatedwith gene expression (Xie et al, 2012). How DNAmethylation of the gene body contributes to gene regulationis still unclear.BASIC MECHANISM OF DNA METHYLATIONThe enzymes that establish, recognize, and remove DNAmethylation are broken into three classes: writers, erasers,and readers. Writers are the enzymes that catalyze theaddition of methyl groups onto cytosine residues. Erasersmodify and remove the methyl group. Readers recognizeand bind to methyl groups to ultimately influence geneexpression. Thanks to the many years of research devoted tounderstanding how the epigenetic landscape is erased andreshaped during embryonic development, many of theproteins and mechanisms involved in DNA methylationhave already been identified.Writing DNA Methylation: the DnmtsThree members of the Dnmt family directly catalyze theaddition of methyl groups onto DNA: Dnmt1, Dnmt3a, and.Neuropsychopharmacology REVIEWS
DNA methylation and its basic functionLD Moore et alREVIEW.4Dnmt3b. Although these enzymes share a similar structurewith a large N-terminal regulatory domain and a C-terminalcatalytic domain, they have unique functions and expression patterns (Yen et al, 1992; Xie et al, 1999). Probably thebest studied Dnmt, especially in the nervous system, isDnmt1, which is highly expressed in mammalian tissuesincluding the brain (Goto et al, 1994). Unlike the otherDnmts, Dnmt1 preferentially methylates hemimethylatedDNA (Pradhan et al, 1999; Ramsahoye et al, 2000). DuringDNA replication, Dnmt1 localizes to the replication forkwhere newly synthesized hemimethylated DNA is formed(Leonhardt et al, 1992). Dnmt1 binds to the newly synthesized DNA and methylates it to precisely mimic theoriginal methylation pattern present before DNA replication(Hermann et al, 2004) (Figure 1b). Additionally, Dnmt1 alsohas the ability to repair DNA methylation (Mortusewiczet al, 2005). For this reason, Dnmt1 is called the maintenance Dnmt because it maintains the original pattern ofDNA methylation in a cell lineage. Knockout of Dnmt1 inmice results in embryonic lethality between E8.0 and E10.5(Li et al, 1992). At this time, knockout embryos exhibita two-thirds loss of DNA methylation, in addition tonumerous apoptotic cells in a variety of developing tissuesincluding the brain. Interestingly, mouse embryonic stemcells lacking Dnmt1 remain viable (Chen et al, 1998).However, in vitro differentiation results in massive celldeath, recapitulating the phenotype observed in knockoutembryos (Jackson-Grusby et al, 2001). These findings firmlyestablish that Dnmt1 plays a critical role in cellulardifferentiation as well as in dividing cells.Dnmt3a and Dnmt3b are extremely similar in structureand function. Unlike Dnmt1, both Dnmt3a and Dnmt3bwhen overexpressed are capable of methylating both nativeand synthetic DNA with no preference for hemimethylatedDNA (Okano et al, 1999). For this reason, Dnmt3a andDnmt3b are referred to as de novo Dnmt because they canintroduce methylation into naked DNA (Figure 1a). Whatprimarily distinguishes Dnmt3a from Dnmt3b is its geneexpression pattern. Although Dnmt3a is expressed relativelyubiquitously, Dnmt3b is poorly expressed by the majority ofdifferentiated tissues with the exception of the thyroid,testes, and bone marrow (Xie et al, 1999). Similar to Dnmt1,the knockout of Dnmt3b in mice is embryonic lethal (Okanoet al, 1999). On the other hand, Dnmt3a knockout mice arerunted but survive to B4 weeks after birth (Okano et al,1999). From these results it appears that Dnmt3b is requiredduring early development, whereas Dnmt3a is required fornormal cellular differentiation.A final member of the Dnmt family is Dnmt3L, a proteinthat lacks the catalytic domain present in other Dnmtenzymes (Aapola et al, 2000; Hata et al, 2002). Dnmt3L ismainly expressed in early development and is restricted tothe germ cells and thymus in adulthood (Aapola et al, 2000,2001). Although Dnmt3L has no catalytic function of itsown, it associates with the Dnmt3a and Dnmt3b andstimulates their methyltransferase activity (Hata et al, 2002;Suetake et al, 2004; Jia et al, 2007). Consistent with its
DNA Methylation and Its Basic Function Lisa D Moore1, Thuc Le1 and Guoping Fan*,1 1Interdepartmental Program in Neuroscience and Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA In the mammalian genome, DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5
DNA cytosine methylation is a major epigenetic mark in eukaryotes. In plants, the DNA methyla-tion level in the genome is controlled by de novo DNA methylation, maintenance DNA methylation and DNA demethylation. De novo methylation is mediated by RNA-directed DNA methylation (RdDM), which can occur at all cytosine contexts,
17% higher DNA methylation levels in exons com-pared to the flanking introns in the equal GC-content group and 5% differential DNA methylation in the other group (Figure 1). Therefore, DNA methylation is more abundant in exons compared to the flanking introns, regardless of the GC-content environment. Thus, DNA methylation can be considered .
orders [8, 11]. Therefore, performing research on DNA methylation plays a critical role in medicine, biological sci-ences, and biochemistry. Given its importance in early development and aging, DNA methylation is a crucial epigenetic modification to profile. The detection of DNA methylation patterns is a
IV. Role of DNA Methylation in the CNS and Neuropsychiatric Disorders V. Conclusion Remarks References DNA methylation is an epigenetic mechanism in which the methyl group is covalently coupled to the C5 position of the cytosine residue of CpG dinucleo-tides. DNA methylation generally leads to gene silencing and is catalyzed by a
methylation results in the conversion of cytosine to 5-methyl cytosine. DNA methylation can lead to changes in gene expression and function without altering the primary sequence of DNA. Methylation can be affected by dietary levels of methyl-donor components, such as folic acid. This may be an important mechanism for environmentally-induced
DNA methylation is a heritable epigenetic modifica-tion found in most eukaryotic organisms including plants, animals, and fungi [12, 13]. In contrast to ani-mals, where DNA methylation occurs mostly at the CG cytosine sequence context, DNA methylation in plants can occur at CG and CHG (where H is A, T, or C) as well as the CHH context [14–16].
the diagnostic and prognostic potential of RASSF1A methylation. This presents RASSF1A methylation as an attractive biomarker for early cancer detection which, for most cancers, results in improved clinical outcome. DNA methylation analysis is applicable to a range of body fluids including serum, urine, bronchioalveolar lavage and sputum.
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